In cardiac muscle, uptake of Ca2+ by mitochondria during the excitation-contraction (EC) coupling is important for synchronizing ATP production with the needs of contraction (excitation-bioenergetics (EB) coupling). However, an integrative mechanism to describe the EB coupling is missing mainly due to the lack of information about the molecular identities of several key proteins involved in this process. Recent ground-breaking studies have shown that mitofusin 2 (Mfn2) is responsible for tethering endoplasmic reticulum to mitochondria. Moreover, several components of the mitochondrial Ca2+ uniporter (mtCU) including its pore unit (MCU) have been uncovered. These progresses open up a new opportunity for applying molecular tools to elucidate the mechanisms of mitochondria-sarcoplasmic reticulum (MITO-SR) tethering in controlling bioenergetics and Ca2+ dynamics. Our labs were the first to show a privileged transport of Ca2+ from SR to mitochondria in cardiomyocytes due to their juxtaposition, secured by tethering with Mfn2 family proteins. Dr. Sheu has a long standing expertise in using genetic and physiological tools to study in and ex vivo the cardiac mitochondrial Ca2+ and reactive oxygen species (ROS) regulation and Dr. Csordas has a strong track record in using biochemical and imaging techniques to investigate MITO-SR tethering and local Ca2+ crosstalk. Together, we will combine these interdisciplinary approaches to test the hypothesis that MITO-SR tethering via Mfn2 family proteins creates a micro-domain of high Ca2+ between these two organelles during EC coupling. Moreover, mtCUs are clustered in the region of inner mitochondrial membrane (IMM) that is in proximity with SR. Losses of this juxtaposition decrease EB coupling efficiency that leads to energy deficiency and oxidative stress and subsequent heart failure (HF). Three specific aims are: 1) to identify the tethering components that bridge MITO-SR associations. Hypothesis: Mfn2, possibly a truncated form, aligns SR with mitochondrial contact points. 2) To determine the distribution of mtCU in the IMM. Hypothesis: mtCU is preferentially localized in the areas where mitochondria and SR are in contact. 3) To elucidate the mechanisms by which the disrupted MITO-SR association leads to HF. Hypothesis: The loss of MITO-SR association leads to the inefficiency of EB coupling, as a result, electron transport chain activities and matrix NADPH levels decrease, which cause ROS to increase. The increase in ROS together with the decrease in ATP enhances the susceptibility of mitochondrial permeability transition pore for opening, especially under the energy-demanding stresses, which leads to cardiac injury and failure. The destruction of mitochondrial Ca2+ homeostasis is a key element for leading to mitochondrial dysfunction-associated clinical phenotypes including heart diseases (e.g. HF), neurodegenerative diseases, metabolic diseases (diabetes), and aging. Because MITO-SR juxtaposition is a critical factor in controlling mitochondrial Ca2+ dynamics, it is of scientific importance and clinical relevance that the present proposal will bring forth the molecular mechanism underlying the cardiac MITO-SR tethering and translate this unique structure to the physiological regulation of mitochondrial Ca2+ influx in bioenergetics and to the pathological implication of energy deficiency and oxidative stress in HF.